Potential Use of Amniotic Membrane - Derived Scaffold for Cerebrospinal Fluid Applications

Scaffolds derived from decellularized tissues provide a natural microenvironment for cell culture. Embryonic cerebrospinal fluid (e-CSF) contains factors which play vital roles in the development of the nervous system. This research was aimed to survey the effect of Wistar rat e-CSF on neural differentiation of bone marrow derived mesenchymal stem cells (BM-MSCs) cultured on the human amniotic membrane (AM). BM-MSCs were collected from femurs and tibias, and were cultured in Dulbecco's Modified Eagle's Medium. The placenta was harvested from healthy women during cesarean section and AM was acellularized using EDTA and physical scrubbing. e- CSF was harvested from rat fetuses at E17. Adequate numbers of BM-MSCs were cultured on acellularized membrane, and were treated with E17 CSF for 7 days. MTT (3-(4, 5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide) assay confirmed the survival and proliferation of BM-MSCs cultured on AM derived scaffold. Hematoxylin/eosin staining and scanning electron microscopy showed the morphological and the structural changes of BM-MSCs throughout the culture and treatment with e-CSF. The results of immunocytochemistry showed that microtubule associated protein 2 and beta-III tubulin were expressed in BM-MSCs cultured on acellular amnion scaffold and treated with e-CSF. Our results showed for the first time that the combination of acellular AM as a natural scaffold and e-CSF as a source of neurological factors could effectively improve the BM-MSCs cultivation and differentiation

he extracellular matrix-based scaffolds act as an excellent three dimensional media for stem cell cultures (1,2). Scaffolds derived from decellularized tissues are a viable alternative to synthetic polymers, and provide a natural microenvironment for cell migration and differentiation (3,4). Amniotic membrane (AM) is a thin, elastic, semi-transparent, and semipermeable fetal derived tissue which is attached to the chorion (5). AM is an easy harvesting tissue and has significant properties such as anti-inflammatory, immunomodulatory, and antimicrobial effects, and is considered as a fascinating biomaterial in the field of tissue T Submitted 8 April 2018; Accepted 14 August 2018; Published 19 August 2018 engineering and regenerative medicine (6). The extracellular matrix of AM contains collagen type IV, type VII, fibronectin, and laminins-1 and -5, and provide a natural scaffold for cell adhesion and proliferation (7).
Cerebrospinal fluid (CSF) bulk of the central nervous system (CNS) comprises the most extracellular fluid of the brain, and fills brain ventricles, subarachnoid space, and the spinal canal (8). CSF is important for the physical protection of a developing brain, and CSF sufficient pressure maintenance in the ventricle is also essential for normal CNS development (9). In addition, CSF circulation applies ''trophic'' effect on the developing brain by providing important growth factors and other biological active substances (10).
In adults, CSF has been considered as an intermediary between blood and brain for transmitting the nutrients and growth factors, and as a liquid buffer to protect the brain and also major vessels that carry the blood to the brain. It was reported that CSF contains nerve growth factor (NGF) and transforming growth factor α (TGF-α) (11). As a whole, the available data indicates the presence of diffusible factors in embryonic CSF (e-CSF) to regulate survival, proliferation, and differentiation of neuroepithelial stem cells (12)(13)(14).
Bone marrow MSC (BM-MSCs) are best characterized and well-established source of MSCs (23); they express and differentiate into three lineage related cells in culture media (24).
Using an in vitro three-dimensional model, we

MTT proliferation assay
To investigate the proliferation rate of BM-MSCs seeded on AM, the 3-(4, 5-dimethylthiazol- intensity of the fluorescent immunocytochemistry images was measured using the Image J software.

Statistical analysis
Quantitative statistical analysis was performed using one-way ANOVA and TUKEY test and significance was accepted for P values of <0.05.

BM-MSCs harvesting and culturing
Bone marrow mesenchymal stem cells were harvested from femur bone of Wistar rat and transferred into culture plates which contained spherical-shaped objects and aggregated cells. After the second passage, debris and non-adherent cells were removed and a homogenous population of BM-MSCs was observed (Fig. 1).

AM scaffold characterization
To confirm complete removing of epithelial cells, hematoxylin-eosin staining was done.
Observations revealed that there were no cell fragments or epithelial cells in decellularized AM (dAM) (Fig. 2). Scanning electron microscopy images confirmed the process of decellularization and dAM had a porous non cellular structure (Fig. 3).

Seeding of BM-MSCs on dAM
Twenty-four hours after AM preparation,   Furthermore, hematoxylin-eosin staining showed the presence of spindle-shaped BM-MSCs on AM scaffold in culture medium (Fig. 4B).    (Fig. 7). The Image J. analysis of fluorescent density revealed that e-CSF promoted the expression of MAP-2 and beta-III tubulin significantly more than b-FGF (Fig. 8).

Discussion
Several studies reported the presence of neurogenic and neurotrophic factors in the CSF, which take part in nervous system development, proliferation, migration, and differentiation of nerve cells (26,27). It was also shown that e-CSF protein structure is much more complex than mature CSF, which include growth factors and cytokines that are effective in cell behavior regulation. One of this neurogenic factors is NGF that has been shown to be present in chick e-CSF, and its level was reported to increase in 17 and 18 embryonic days (28,29). Laboratory findings showed that change and, more importantly, loss of e-CSF strongly reduce nerve cells populations in the nervous system (9). In general, CSF has a key role as a liquid path for soluble signals transmission, and in this manner, it affects brain parenchyma cells behavior (30). The neural differentiation of BM-MSCs has been analyzed by different researchers, either in conventional two-dimensional (2D) culture (35,36) or three-dimensional (3D) culture (37)(38)(39). In the current study, AM-derived scaffold was used to 3D culture of BM-MSCs. For AM decellularization, and blocking the activity of proteases during cell lysis, EDTA containing trypsin was used in our procedure. In this decellularization process, collagen, elastin, and glycosaminoglycans are not damaged (40,41), and the interconnected pore networks within the scaffold are kept to provide the spatial position of the interactions of cytokines and growth factors released from cells (42,43).
According to the scanning electron microscope, acellular amniotic scaffold pore size